Institut Cochin, Département d'Immunologie-Hématologie, Paris, France.
PLoS One. 2013 Apr 23;8(4):e60961. doi: 10.1371/journal.pone.0060961. Print 2013.
The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.
干细胞因子受体 (SCF) c-Kit 在调节多种细胞类型的细胞增殖和存活中起着关键作用。特别是，c-Kit 是早期红系祖细胞扩增所必需的，而当细胞以依赖促红细胞生成素的方式进入成熟的最后步骤时，它必须从细胞表面消失。我们最初观察到，伊马替尼 (IM)，一种针对 c-Kit 酪氨酸激酶活性的抑制剂，同时下调 c-Kit 的表达，并在没有 SCF 的情况下加速促红细胞生成素驱动的红细胞分化。我们研究了 IM 或相关马替尼 (MA) 在缺乏 SCF 的情况下诱导 c-Kit 下调的机制在人类 UT-7/Epo 细胞系中。我们发现，在 IM 或 MA 存在下，c-Kit 的下调被预先孵育甲基-β-环糊精抑制，表明 c-Kit 在没有配体的情况下被内化。与 SCF 不同，TKI 诱导的内化不依赖于 E3 泛素连接酶 c-Cbl。此外，c-Kit 通过溶酶体而不是蛋白酶体途径降解。在脉冲追踪实验中，IM 不会调节 c-Kit 的合成或成熟。在未用 IM 处理或用 IM 处理的 UT-7/Epo 细胞中分析磷酸酪氨酸肽表明，IM 不会改变这些细胞中的整体酪氨酸磷酸化。此外，我们表明，阻止 IM 完全进入 ATP 结合口袋的 T670I 突变，在 IM 存在的情况下不允许内化过程。总之，这些数据表明，TKI 诱导的 c-Kit 内化与 ATP 结合口袋完整性的改变有关。
